Free-Living Amoeba act as transient permissive hosts for Leptospira spp.

BackgroundLeptospirosis is a zoonotic disease caused by pathogenic Leptospira spp., which persist in soil and water environments for extended periods of time. The mechanisms enabling this environmental survival remain elusive. Free-living amoebae (FLA) are widespread protozoa that act as reservoirs or "Trojan horses" for numerous bacterial pathogens, protecting them from stress and contributing to their persistence. Whether pathogenic Leptospira exploit similar interactions with FLA has not been resolved. Methodology/Principal FindingsUsing live confocal microscopy, flow cytometry, and gentamicin protection assays, we investigated the interactions between pathogenic (Leptospira interrogans) and saprophytic (Leptospira biflexa) leptospires with three FLA species: Acanthamoeba castellanii, Dictyostelium discoideum, and Hartmannella vermiformis. While rapid internalization was observed, entry was only partially dependent on actin-driven processes and was enhanced by the presence of live bacteria. Following internalization, bacteria persisted for at least 48h as indicated by colony-forming assays. However, no evidence of intracellular replication was detected. The number of fluorescently labeled leptospires progressively declined over time, providing further evidence of leptospires survival without multiplication. Finally, analysis of environmental soils in New Caledonia showed co-occurrence of FLA and Leptospira. Soil-derived FLA also internalized pathogenic Leptospira in vitro, showing that these interactions extend to natural isolates. Conclusions/SignificanceOur results demonstrate that free-living amoebae internalize both pathogenic and saprophytic leptospires and allow their transient persistence without replication. By providing protection and prolonging viability in soil environments, FLA may contribute to the ecological maintenance of Leptospira. These findings pinpoint FLA as potential environmental reservoirs that could play a role in shaping leptospires survival strategies relevant for transmission and host infection. Author SummaryFor bacteria living in soils and freshwater environments, survival depends on their ability to adapt to complex ecological landscapes populated by numerous predators and competitors. In such habitats, interactions with other microorganisms are unavoidable and may shape long-term survival strategies. Pathogenic Leptospira, the bacteria responsible for leptospirosis, can persist for long periods outside their hosts, yet the ecological mechanisms supporting this environmental survival remain poorly understood. In soil and freshwater ecosystems, microscopic predators known as free-living amoebae commonly feed on bacteria. However, several bacterial pathogens can survive inside these amoebae and use them as temporary shelters. Because ancestral Leptospira were soil-dwelling saprophytes, interactions with amoebae likely represent an ancient ecological relationship in which successful survival strategies may have evolved and remain conserved in present-day pathogenic species. With this perspective in mind, we used microscopy approaches and bacterial viability assays to investigate whether Leptospira interacts with amoebae. We found that several amoeba species rapidly engulf both pathogenic and non-pathogenic Leptospira. Once internalized, the bacteria remained viable for up to two days but did not multiply. We also detected both amoebae and Leptospira in the same soil samples and showed that environmental amoebae could internalize the bacteria. These findings suggest that amoebae may act as temporary shelters for Leptospira, helping them persist in soils and water and potentially contributing to the environmental stage of leptospirosis transmission.